WO2006058439A1 - Appareils et procédés de dépôt de gouttelettes de liquide - Google Patents

Appareils et procédés de dépôt de gouttelettes de liquide Download PDF

Info

Publication number
WO2006058439A1
WO2006058439A1 PCT/CA2005/001843 CA2005001843W WO2006058439A1 WO 2006058439 A1 WO2006058439 A1 WO 2006058439A1 CA 2005001843 W CA2005001843 W CA 2005001843W WO 2006058439 A1 WO2006058439 A1 WO 2006058439A1
Authority
WO
WIPO (PCT)
Prior art keywords
capillary
plate
droplet
tip
capillary tube
Prior art date
Application number
PCT/CA2005/001843
Other languages
English (en)
Inventor
Liang Li
J. Bryce Young
Original Assignee
The Governors Of The University Of Alberta
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Governors Of The University Of Alberta filed Critical The Governors Of The University Of Alberta
Priority to US11/720,699 priority Critical patent/US8202494B2/en
Publication of WO2006058439A1 publication Critical patent/WO2006058439A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/84—Preparation of the fraction to be distributed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1065—Multiple transfer devices
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0409—Sample holders or containers
    • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0431—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for liquid samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/84—Preparation of the fraction to be distributed
    • G01N2030/8411—Intermediate storage of effluent, including condensation on surface
    • G01N2030/8417—Intermediate storage of effluent, including condensation on surface the store moving as a whole, e.g. moving wire
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1034—Transferring microquantities of liquid
    • G01N2035/1039—Micropipettes, e.g. microcapillary tubes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/80—Fraction collectors
    • G01N30/82—Automatic means therefor

Definitions

  • This invention relates to mass spectroscopy, and more particularly this invention relates to the preparation of samples for use in matrix assisted laser desorption ionization (MALDI) mass spectroscopy.
  • MALDI matrix assisted laser desorption ionization
  • Electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are complementary ionization techniques for generating ions for mass spectrometry (MS) in the field of proteomics.
  • Liquid chromatography (LC) is typically interfaced to ESI-MS instruments to give online sample separation and MS analysis.
  • MALDI demonstrates marked robustness towards contaminants, and features greater analysis speed and reduced sample consumption.
  • MALDI is not amenable to a direct interface with LC, and thus the sample separation power of chromatography is not easily available to MALDI-MS.
  • LC-MALDI refers to techniques that attempt to bridge the gap between LC and MALDI.
  • the invention can be useful to other areas of applications such as in tissue imaging by MALDI.
  • Tissue imaging by MALDI involves producing thin slices of tissue, and directly or indirectly using MALDI-MS to create a mass spectral map of the tissue, based on the chemical/protein environment. Where this invention is applicable is in the deposition of a MALDI matrix at discrete locations on the tissue slice.
  • the technique requires the use of a conductive receiving plate, precluding it from more general use, such as sample or matrix deposition to a non-conductor such as a glass slide. It involves high voltage (2000-4000 V) which can make the device more expensive to construct, and introduces special precautions which must be taken during operation.
  • an apparatus for generating a fraction from a liquid sample comprising:
  • a stop member located to limit the travel of the moveable element, whereby, in use, actuation of the actuation means displaces a moveable element until the moveable element abuts the stop member, to rapidly decelerate the moveable element and the capillary tube, to promote the separation of a droplet from the tip of the capillary tube.
  • the capillary tube can be mounted within a support tube.
  • any suitable actuation means can be used, but it preferably comprises a solenoid and a solenoid core mounted on the support tube. The locations of the solenoid and the core can be reversed.
  • the actuation means can include a spring means, acting on the moveable element, to displace the moveable element to a normal position.
  • the stop member can be mounted on the support tube and can be generally cylindrical, but it is to be understood that it is not limited to such a configuration.
  • a fixed abutment member can be located adjacent the stop member and can include an aperture for passage of the support tube and the capillary tube.
  • the apparatus can include means for supporting a support plate for receiving droplets adjacent the capillary tip.
  • the apparatus can include a means for locating the support plate at a first position sufficiently spaced from the capillary tip to promote complete separation of larger droplets from the capillary tip before the droplets contact the plate, and a second position in which the plate is located closer to the capillary tube, for enabling smaller droplets to be drawn onto the plate by wicking action for complete separation of the droplets from the capillary tip.
  • the apparatus can include a support member and a plurality of capillary tubes, each including a respective capillary tip mounted in the support member, the tips of the capillary tubes being mounted in a generally common plane, for enabling simultaneously deposition of a plurality of fractions onto a plate.
  • the capillary tip can be coated with a hydrophobic coating.
  • Another aspect of the present invention provides a method of generating a fraction from a liquid sample for analysis, the method comprising:
  • Rapid deceleration of the capillary tube can be effected by means of impact against an abutment member.
  • the method comprises, to form large droplets, mounting the capillary tube sufficiently spaced from the plate, to cause droplets to be entirely separated from the capillary tube before contacting the plate.
  • the method comprises, to form small droplets, mounting the capillary tip sufficiently close to the plate whereby a droplet at the capillary tip extends and elongates and contacts the plate before complete separation from the capillary tip, whereby deposition of the droplet on the plate is promoted by a wicking action.
  • the method can include adding matrix material to the liquid solution, prior to supply to the capillary tube.
  • the method can also include mounting a plurality of capillary tubes on a common support member, supplying each of the capillary tubes with a liquid sample, and simultaneously subjecting the support member and all the capillary tubes to rapid deceleration, to deposit a plurality of droplets as fractions, simultaneously, onto the plate.
  • the method can include providing a tissue sample on the plate and providing at least one droplet on the tissue sample.
  • Figure 1 shows, schematically, an apparatus in accordance with a first embodiment of the present invention
  • Figure 2 shows use of the apparatus of Figure 1 to generate a relatively large drop
  • Figures 3a-3d show an apparatus in accordance with a second embodiment of the present invention, and a sequence for generating or transferring and transferring a smaller droplet;
  • Figure 4 is a perspective view showing use of the apparatus of the present invention to provide a two-dimensional array of droplets on a MALDI plate;
  • Figure 5 shows a variant of the apparatus of Figure 4, including the ability to multiplex different samples and generate the two-dimensional array on the MALDI plate;
  • Figure 6 shows use of the apparatus of the present invention to provide a two-dimensional array of droplets on a tissue sample, in accordance with the present invention
  • Figure 7 shows a variant of the apparatus of Figure 6, including multiplexing a number of samples and generating a two-dimensional array on a tissue sample;
  • Figure 8 shows a formation of MALDI spots on a plate from post-column addition of matrix to LC-MALDI fractions;
  • Figures 9-11 show spectra generated from MALDI samples formed in accordance with the present invention.
  • Figure 12A is a Mass spectrum of 5 fmol pyro-GFP generated by LC-MALDI MS;
  • Figure 12B is a Mass spectrum of 5 fmol pyro-GFP generated direct MALDI-TOF MS;
  • Figure 13A-13F are LC-MALDI mass spectra of A) fraction 28 and B) fraction 29 of a 200-fmol BSA digest; C) and E) LC-MALDI MS/MS spectra of L421-R433 5fmol and 200 fmol respectively and D) and F) R360- R371 from 5 fmol and 200 fmol BSA digest;
  • Figure 14 is a sequence coverage of BSA by LC-MALDI MS/MS of the digests using different amounts of sample loading;
  • Figure 15A is a LC-MALDI mass analysis of an E. coli protein extract digest in which 25 fractions collected at 60 second intervals result in the detection of 145 unique peptides;
  • Figure 15B is a LC-MALDI mass analysis of an E. coli protein extract digest in which 100 fractions collected at 15 second intervals result in the detection of 409 unique peptides. DESCRIPTION OF VARIOUS EMBODIMENTS
  • the apparatus of the present invention is indicated generally by the reference 10, having a capillary tube 12, that has an inlet end 14, for connection to a liquid supply and an outlet end or tip 16, providing a tip for formation of droplets.
  • the capillary tube 12 is mounted in a support tube 18, here a metal tube, to provide the necessary constructional integrity to the capillary tube 12.
  • the metal tube 12 is itself mounted in a solenoid core 20.
  • a stop member 22 is mounted on a lower end of the metal tube 18.
  • a solenoid for actuating the apparatus is indicated at 30 and is mounted with respect to a support 32.
  • a plate 34 is mounted across the bottom of the support 32 and includes an aperture 36, through which the capillary and metal tube can extend.
  • the aperture 36 is sized so that the stop member 22, when traveling downwards, abuts the plate 36 and is stopped.
  • the plate 34 does not necessarily have to be in the form of a plate and it is sufficient if there are one or more projections to engage and stop the stop member 22.
  • the capillary tube 12, metal tube 18, solenoid core 20 and stop member 22 comprise a moveable element 24 that, as will be detailed, can be displaced downwards by the action of the solenoid 30.
  • a coil spring 38 is provided around the metal tube 18, below the solenoid core 20, and abutting an inwardly projecting lip 40 at the bottom of the solenoid 30.
  • the inwardly projecting lip 40 can be in the form of an aperture or a hole in a plate, or it can be in the form of one or more projections sufficient to support the bottom end of the spring 38. Accordingly, Figure 1 shows the normal position of the device, when the solenoid 30 is not activated.
  • a small DC voltage is applied over a very short time period (typically 0.10sec) to actuate the solenoid.
  • the shape of the voltage pulse is a square wave, although other wave shapes are possible. This generates a downward force on the moveable element 24 accelerating it downwards towards the plate 34. The element 24 accelerates to a maximum velocity, until the metal stop member 22 contacts the plate 34. This contact creates an impact rapidly decelerating the moveable element 24 to a stopped position.
  • the apparatus 10 is mounted above a plate 50 for receiving one or more sample droplets, and the apparatus 10, preferably, is integral with a table for supporting the plate 50 at a desired height relative to the outlet and or tip 16 of the capillary 12. As is explained further below, the spacing between the tip of the capillary 12 and the plate 50 can be varied, depending upon the size of the sample droplets.
  • liquid sample is supplied to the inlet end 14 of the capillary 12. As the liquid sample is supplied, a droplet will start to form at the capillary tip 16, as indicated at 52.
  • a sudden impact of the stop member 22 against the plate 34 generates an impact force that dislodges the droplet 52 from the capillary tip 16.
  • the tip may be coated with a hydrophobic coating to reduce droplet adhesion to the capillary.
  • Figure 1 shows the basic configuration of a first embodiment of the apparatus adapted for generating relatively large droplets, greater than 100nl, and for this purpose the capillary tip 16 is located a relatively large distance above the plate 50.
  • Figure 2 shows operation of this first embodiment of the apparatus.
  • Figures 3a-3d show a second embodiment of the apparatus, that corresponds generally with the first embodiment of Figures 1 and 2, so that like components are given the same reference numeral.
  • a shown in Figure 3a the principal difference is that the tip 16 of the capillary is now closer to the plate 50, to enable smaller droplets, less than 10OnI, to be formed and to be transferred by a modified technique as detailed below.
  • Figure 3b shows a droplet 54 just as the impact commences, before the severe decelerating affects of the impact have distorted the droplet, and it will be understood that, in all these figures, the droplet is, to a large extent, shown schematically.
  • the droplet 54 is shown elongating and extending downwards from the capillary tip 16 due to the decelerating effect of the impact, until it contacts the plate 50. This enables the droplet to transfer by a wicking action and does not require the droplet to separate from the tip 16 and transfer by momentum to the plate 50.
  • the droplet 54 is shown detached from the capillary tip 16, so as to be deposited on the plate 50, as indicated at 54a.
  • the separation of the capillary tip in both positions is somewhat arbitrary, but is related to the type of fluid (and the size of the fluid droplet) being deposited.
  • the separation between capillary tip and plate surface is, usually, several millimeters.
  • the separation is, usually, several hundred microns (micrometers) to a millimeter.
  • FIG 4 shows the apparatus 10 used in conjunction with a plate 60 enabling a two-dimensional array of sample spots to be deposited on it.
  • the plate 60 is mounted on a two-dimensional translational stage.
  • the two-dimensional stage is driven, and moves the plate 60 in the manner of raster, in the two dimensions, to generate a two- dimensional array of spots.
  • the plate 60 is moved in a predefined path to predefined positions to collect fractions from the deposition apparatus 10.
  • FIG. 5 shows a variant of the arrangement shown in Figure 4, adapted for multiplexing, for parallel depositions.
  • the capillary 12 through the center of the solenoid is omitted.
  • the tube 18 is replaced with a rod 64 that extends down through the solenoid 30. Attached to the bottom of the rod 64 is a cross member 66. It will be understood that this arrangement is shown purely schematically, and the mechanical details will be such as to ensure that each of the capillaries, detailed below, is subject to the same impact and sudden deceleration effect.
  • a plurality of capillaries 68 are connected through the cross member 66. With four capillaries, for each actuation of the apparatus 62, four spots are deposited on the plates 60 in a predetermined spacing.
  • the collected fractions after drying can be inserted into a
  • MALDI-MS for analysis.
  • Another technique commonly used is analysis of tissue.
  • a plate 70 has a thin slice of tissue sample indicated schematically 72 placed upon it.
  • the matrix spots are deposited by a hand-operated pipet.
  • the matrix spots each promote the generation of ions from the tissue sample at that particular spot.
  • the apparatus 10 can be used to deposit hundreds of thousands of spots on the tissue sample in a relatively short period of time.
  • the use of a two-dimensional translational stage engages these spots to be located accurately on the desired grid pattern.
  • the apparatus 10 enables enable fine control over both spot size and spot placement, affording significant gains over known manual techniques.
  • Figure 7 shows the use of the multiplexing apparatus 62 of
  • Figure 5 employed with a tissue sample.
  • the present invention can also be used in the analysis of small molecules such as organic pharmaceuticals.
  • the device can be used to deposit sample on the mounting plate, and again, for parallel applications, the apparatus of Figure 5 can be used to multiplex the samples and deposit multiple spots simultaneously.
  • this shows of the appearance of MALDI spots on a sample plate, after the fractions have been deposited on the plate.
  • fractions are taken from a liquid chromatograph (LC), and matrix is added to the liquid as it elutes from the column.
  • LC liquid chromatograph
  • Figures 9, 10 and 11 show MALDI spectra obtained using the present invention. These demonstrate that the device functions as intended. Since the device serves to fractionate a flowing liquid stream, into many small fractions, it can give improved resolution. The flowing liquid stream can contain many discrete components that elute from the exit of the capillary at different times. Collecting shorter fractions affords better resolution, allowing one to focus on fewer components in any given fraction. Collecting longer fractions may maximize the amount of one particular component in a given fraction. This maximizes the probability of detection using a mass spectrometer.
  • Figure 9 shows how a very long fraction can be used to detect a component that is present only in very small amounts.
  • Figures 10 and 11 are actually a series of two fractions, intended to demonstrate the chromatographic resolution of a very complex mixture. Some of the peaks in each mass spectrum are common, indicating that some components are eluting during the collection of both fractions.
  • the droplet may be entirely separated from the tip of the capillary, before contacting the plate on which it is to be deposited; for smaller droplets, the configuration can be such as to promote elongation of the droplet, so that it contacts a surface and a wicking action then causes the droplet to be absorbed onto the surface.
  • HPLC grade acetonitrile (ACN) and methanol (MeOH) were obtained from Fisher Chemicals (Fairlawn, NJ), and filtered prior to use.
  • Water (H 2 O) was deionized with a Millipore deionizer to 18 MOhm and filtered through a 22 ⁇ m filter prior to use.
  • Dithiothreitol (DTT), iodoacetamide (IAA), trifluroacetic acid (TFA), bovine serum albumin (BSA), trypsin enzyme, and pyro-Glu-fibrinopeptide (pyro-GFP) were obtained from Sigma Aldrich (St. Louis, MO) at the highest available purity.
  • DHB 2,5- dihydroxybenzoic acid
  • E. coli Protein Extraction E. coli extracts were prepared by a solvent suspension method. Lyophilized E. coli cells (6 mg) were suspended in 2 mL of 10 mM Tris-HCI buffer (pH 7.90) in a centrifuge tube and sonicated for 1 min with a probe tip sonicator (Branson Sonifier 450, Branson Ultrasonics, Danbury, CT) while the centrifuge tube was immersed in an ice bath. The suspension was centrifuged at 1175Og for 10 min.
  • the supernatant was transferred in 500 ⁇ L aliquots to Microcon-3 3000 Da molecular weight cutoff filters (Millipore, Billerica, MA) and centrifuged at 1300Og for 30 min. The filtrate was collected, pooled, and the protein content quantified by Bradford assay, using ⁇ -globulin as a protein standard.
  • An Agilent 1100 Capillary HPLC system (Palo Alto, CA) was used to perform the reversed phase separation on a 150 ⁇ m x 15 cm C18 column (Grace Vydac, Hesperia, CA).
  • a reproducible flow rate of 1 ⁇ L/min through the column was achieved by using a chromatographic splitter, installed upstream of the sample injector.
  • the pump was operated at a flow rate of 100 ⁇ L/min, and the split ratio was adjusted to approximately 100:1.
  • the flow rate was subsequently tested using volumetric glass capillaries.
  • the mobile phase gradient was generated using a binary mixture of A: 0.1% TFA in 4% ACN/H 2 O, and B: 0.1 % TFA in ACN.
  • the gradient program used was 0% B for 5 minutes, 0-15% B from 5 to 7.5 min, 15-25% B from 7.5 to 25 min, 25-35% B from 25 to 35 min, and 35-80% B from 35 to 55 min.
  • FIG. 1A A diagram of the deposition system is shown in Figure 1A.
  • the micro-depositor consists of a small solenoid coil (TP6X12-I-24D, Guardian Electric, Woodstock, IL) which is mounted in place with a homebuilt cradle/motion control system.
  • the solenoid core is a homebuilt hollow ferromagnetic steel dowel, with a custom fitted steel capillary.
  • a fused silica capillary i.d. 50 ⁇ m and o.d. 180 ⁇ m
  • the solenoid assembly is mounted on a x,y,z micro-adjustment stage (1.75" three axis centre drive positioning stage, Edmund Optics, Barrington, NJ).
  • the assembly is positioned above motorized translation tables, as shown in Figure 1 B (MX80S, Parker Hannifin, Rohnert Park, CA).
  • the motor drivers, computer control card, breakout board and cables required for the motion tables were acquired from the manufacturer.
  • the solenoid is operated by a homebuilt power supply with an internal function generator which produces a square wave.
  • the power supply can be remotely toggled via a gated logic signal, which is triggered through the break-out board.
  • the motion tables and solenoid power supply are controlled by a program that was written using the supplied software development tools (ACR-View, Parker Hannifin Compumotor Division, Rohnert Park, CA).
  • Matrix Addition Matrix was added during the deposition via a post-column T-connection (VICI ⁇ /alco, Houston, TX). DHB solution was infused into the T-connection using a syringe pump (Cole-Parmer, Vernon Hills, IL) at a flow rate of 1 ⁇ L/min. The LC fractions were deposited on a 100- spot MALDI target (Applied Biosystems, Foster City, CA) that was fixed into position on the motion tables by a homebuilt mounting adapter.
  • MALDI MS data were acquired either using a MALDI time-of-flight (TOF) mass spectrometer (Bruker Reflex, Bremen, Germany) in a reflectron mode, or using the Sciex Qstar Pulsar-i Quadrupole- TOF (QqTOF) mass spectrometer (Applied Biosystems, Concord, ON). MS/MS spectra were collected using the Qstar instrument.
  • the Information Dependent Acquisition (IDA) system was used to automatically and independently acquire MS/MS data.
  • the orthogonal MALDI source came equipped with sampling algorithms that were used to automatically target the laser on the samples deposited on the MALDI plate.
  • the impulse-driven momentum transfer depositor is capable of depositing droplets of both small and large volumes.
  • the typical flow rate is 1 ⁇ L/min.
  • the matrix solution is infused post-column at 1 ⁇ L/min, or 1 :1 with the HPLC.
  • droplet depositions are typically made every 5 to 60 s.
  • the volume of the deposited droplets ranges between 160 nl_ to 2 ⁇ l_.
  • a 2 ⁇ l_ droplet typically dries to approximately 2 mm in diameter. Since in this example the matrix is infused post-column, matrix spots crystallize on the target once the carrier solvents evaporate. Droplets of smaller volume in the droplet range between 160 nl_ to 2 ⁇ l_ are advantageous in this case, since the droplets will evaporate more quickly, leading to smaller overall sample spots. Generating smaller spots tends to produce fewer "hot spots" of signal intensity during MS analysis, thus facilitating the automation of data acquisition.
  • different matrices can be used to alleviate the occurrence of hot-spots, most notably with ⁇ - cyano-4-hydroxycinnaminic acid (CHCA).
  • CHCA ⁇ - cyano-4-hydroxycinnaminic acid
  • CHCA is a "hot" matrix, which can preclude its use in certain MALDI experiments, such as in the analysis of phosphopeptides, due to in-source fragmentation and metastable ion formation.
  • the chromatographic separation can be represented by a continuous stream deposited on the collection plate or as discrete spots.
  • a continuous stream would ideally provide superior chromatographic resolution, although in practice, migration of sample components along the continuous stream may broaden chromatographic peaks.
  • Collecting the eluent as discrete spots at a sufficient frequency can preserve the chromatographic integrity of the separation, and serve to concentrate minor components to small areas for improved detection.
  • producing smaller fractions at greater frequency better preserves the chromatographic separation of components. Larger fractions produced at lower frequency sacrifices separation resolution in favor of greater sensitivity, providing that ion suppression is not severe in both cases.
  • each of these scenarios presents unique advantages, and must be chosen to reflect the needs of the experiment.
  • MALDI interface was demonstrated by fractionating tryptic digests of BSA. Different amounts of a BSA digest were analyzed to gauge the sequence coverage and detection sensitivity. The results are shown in Figures 13 and 14. In all cases, chromatographic base-peak widths for peptides from the reversed-phase separation were approximately 40 seconds. For the best sensitivity of detection, fractions of the separation were collected every 60 seconds to reduce the incidence of peak splitting between fractions. Each separation was collected over the entire course of the 50 minutes gradient in one minute fractions, giving 50 discrete fractions. The fractions were each analyzed by MALDI MS/MS. The analysis was automated using the MALDI spot raster and data-dependent subroutines supplied with the instrument. This was done to both reduce the level of user intervention, and to better reflect the current state-of-the art for MALDI MS/MS using the QqTOF instrument.
  • Figures 13A and 13B show MS spectra obtained from two adjacent, representative fractions, 28 min and 29 min, in an LC-MALDI experiment using 200 fmol of the BSA digest. Fractions 28 min and 29 min are representative of the fractions collected for this BSA digest separation, showing only a handful of unique masses in each fraction. The masses identified in each fraction were subjected to MS/MS analysis.
  • Figures 13(C-F) show MS/MS spectra from two different BSA digest sample loading amounts, 5 and 200 fmol. Two peptides, L421-R433 and R360-R371 , are represented in both samples. The spectra illustrate that even in the 5 fmol sample, the MS/MS data contain enough product fragment ions to identify the peptide using a database search.
  • the BSA tryptic digest was fractionated and deposited at several sample loading amounts, between 5 fmol and 1 pmol. The resultant
  • MS/MS data were searched using the MASCOT algorithm on the Swissprot database. Characteristic peptides of BSA were identified at all sample loading amounts, and sequence maps were produced to illustrate the change in sequence coverage relative to peptide concentration ( Figure 14). Using the smallest sample amount, 5 fmol, 6% of the sequence is accounted for, with 3 characteristic peptides identified. Thus, the LC-MALDI MS/MS technique using the impulse depositor appears to be applicable to analyze proteome samples containing low fmol of proteins. As expected, to increase sequence coverage, a larger amount of sample is required. Using 1 pmol of BSA digest, sequence coverage of 57% can be achieved, as shown in Figure 6.
  • Figure 14 also illustrates that the collection time for each fraction is sufficient for samples of this complexity.
  • Two experiments were performed at the 200 fmol level: BSA digest was fractionated using 30 s intervals, and again at 60 s intervals. The sequence coverage is approximately equal between the two, indicating that, for this simple protein digest, the analyses of the fractionated peptides are not significantly affected by signal suppression and the spectral recording duty cycle of the mass spectrometer.
  • FIG. 15 presents the data as m/z vs. time plots, illustrating the difference in the number of detected peptides. It can be seen from Figures 15A and 15B that, for very complex samples such as this E. coli protein extract, shorter fraction intervals allow for better MS detection, yielding a more complete analysis of the components within the sample. For most peptides detected using the 60 s fraction collection, they were observed in a single fraction. In the case of 15 s fraction collection, some peptides were detected in multiple spots. This is due to the splitting of the analyte peak over several spots.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Optics & Photonics (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

L’invention concerne un procédé et un appareil permettant de générer des gouttelettes, avec un tube capillaire monté dans un élément mobile. Le tube capillaire est accéléré vers une plaque, puis soumis à une rapide décélération, par exemple à l’aide d’un impact, pour favoriser la séparation d’une gouttelette que l’on va ensuite déposer sur la plaque. La gouttelette peut être entièrement séparée de la pointe capillaire avant de se déposer, ou bien, pour des gouttelettes plus petites, la gouttelette s’étend tout simplement de la pointe capillaire, avant d’être étirée sur la plaque par un effet de mèche. La pluralité de tubes capillaires peut être montée sur l’élément de support commun pour effectuer le multiplexage de gouttelettes.
PCT/CA2005/001843 2004-12-02 2005-12-02 Appareils et procédés de dépôt de gouttelettes de liquide WO2006058439A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US11/720,699 US8202494B2 (en) 2004-12-02 2005-12-02 Apparatus and methods for liquid droplet deposition

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US63226604P 2004-12-02 2004-12-02
US60/632,266 2004-12-02

Publications (1)

Publication Number Publication Date
WO2006058439A1 true WO2006058439A1 (fr) 2006-06-08

Family

ID=36564730

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2005/001843 WO2006058439A1 (fr) 2004-12-02 2005-12-02 Appareils et procédés de dépôt de gouttelettes de liquide

Country Status (2)

Country Link
US (1) US8202494B2 (fr)
WO (1) WO2006058439A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2546407A (en) * 2015-12-22 2017-07-19 Micromass Ltd Secondary Ultrasonic Nebulisation

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5072682B2 (ja) * 2008-03-28 2012-11-14 富士フイルム株式会社 質量分析用デバイス、これを用いる質量分析装置および質量分析方法
DE112015001328B4 (de) 2014-03-18 2020-06-04 Micromass Uk Limited Matrixunterstützte Flüssigextraktions-Laserdesorptionsionisations-Ionenquelle

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5957167A (en) * 1997-12-18 1999-09-28 Pharmacopeia, Inc. Article for dispensing small volumes of liquid
US6116197A (en) * 1996-12-19 2000-09-12 Honda Giken Kogyo Kabushiki Kaisha Vertical internal combustion engine
GB2375315A (en) * 1998-11-04 2002-11-13 Biorobotics Ltd Dropping pin non-contact liquid transfer method
CA2451314A1 (fr) * 2001-06-20 2003-01-16 Cytonome, Inc. Systeme de distribution de gouttelettes
US6551557B1 (en) * 1998-07-07 2003-04-22 Cartesian Technologies, Inc. Tip design and random access array for microfluidic transfer
US20050072218A1 (en) * 2003-09-24 2005-04-07 Shimadzu Corporation Fractionating apparatus

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6116197A (en) * 1996-12-19 2000-09-12 Honda Giken Kogyo Kabushiki Kaisha Vertical internal combustion engine
US5957167A (en) * 1997-12-18 1999-09-28 Pharmacopeia, Inc. Article for dispensing small volumes of liquid
US6551557B1 (en) * 1998-07-07 2003-04-22 Cartesian Technologies, Inc. Tip design and random access array for microfluidic transfer
GB2375315A (en) * 1998-11-04 2002-11-13 Biorobotics Ltd Dropping pin non-contact liquid transfer method
CA2451314A1 (fr) * 2001-06-20 2003-01-16 Cytonome, Inc. Systeme de distribution de gouttelettes
US20050072218A1 (en) * 2003-09-24 2005-04-07 Shimadzu Corporation Fractionating apparatus

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2546407A (en) * 2015-12-22 2017-07-19 Micromass Ltd Secondary Ultrasonic Nebulisation
GB2546407B (en) * 2015-12-22 2020-04-08 Micromass Ltd Secondary Ultrasonic Nebulisation

Also Published As

Publication number Publication date
US8202494B2 (en) 2012-06-19
US20090302208A1 (en) 2009-12-10

Similar Documents

Publication Publication Date Title
US20060266941A1 (en) Method and apparatus for interfacing separations techniques to MALDI-TOF mass spectrometry
Ackermann et al. Recent advances in use of LC/MS/MS for quantitative high-throughput bioanalytical support of drug discovery
Niessen State-of-the-art in liquid chromatography–mass spectrometry
Tomer Separations combined with mass spectrometry
US20020139751A1 (en) Microchip electrospray device and column with affinity adsorbents and use of the same
Foret et al. Liquid phase interfacing and miniaturization in matrix‐assisted laser desorption/ionization mass spectrometry
Wickremsinhe et al. A review of nanoelectrospray ionization applications for drug metabolism and pharmacokinetics
Yang et al. Recent technical progress in sample preparation and liquid-phase separation-mass spectrometry for proteomic analysis of mass-limited samples
WO2003042233A2 (fr) Procede de surveillance de la production et de la purification de polypeptides par spectrometrie de masse a desorption/ionisation de surface par impact laser amelioree
Peš et al. Off-line coupling of microcolumn separations to desorption mass spectrometry
CA2518706A1 (fr) Methode et appareil pour connecter un dispositif d'alimentation en analyte a un dispositif de traitement electrodynamique de gouttelettes
US8202494B2 (en) Apparatus and methods for liquid droplet deposition
Miliotis et al. Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry
EP1550862B1 (fr) Procédé d'identification d'un analyte par électrophorèse sur gel et spectrometrie de masse
Young et al. An impulse-driven liquid-droplet deposition interface for combining LC with MALDI MS and MS/MS
Mueller et al. LC-MALDI MS and MS/MS—an efficient tool in proteome analysis
US20030180957A1 (en) Target and method
US20050269507A1 (en) Frontal affinity chromatography/MALDI tandem mass spectrometry
Hioki et al. Nano-LC/MALDI-MS using a column-integrated spotting probe for analysis of complex biomolecule samples
US20030141253A1 (en) Apparatus for efficient liquid chromatography/mass spectrometry processing
Edwards et al. Hyphenating liquid phase separation techniques with mass spectrometry: on-line or off-line
GB2396314A (en) Separating bio-substances for mass spectrometric analysis
Williams et al. Disposable chromatography for a high-throughput nano-ESI/MS and nano-ESI/MS-MS platform
Bereszczak et al. Offline and online liquid chromatography mass spectrometry in quantitative proteomics
CA2566815A1 (fr) Chromatographie d'affinite frontale/spectrometrie de masse en tandem maldi

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05819435

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 11720699

Country of ref document: US